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Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer.

Chen P, Hu T, Liang Y, Jiang Y, Pan Y, Li C, Zhang P, Wei D, Li P, Jeong LS, Chu Y, Qi H, Yang M, Hoffman RM, Dong Z, Jia L - Oncotarget (2015)

Bottom Line: However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance.Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins.Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT
Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.

No MeSH data available.


Related in: MedlinePlus

Production of excessive ROS contributes to MLN4924+CQ-induced NOXA transactivation and apoptotic induction of liver-cancer cells(A) Effect of MLN4924 or CQ alone or the MLN4924+CQ combination, on ROS generation. HepG2 and Huh7 cells were pre-treated with NAC (50 μM) for 2 hours. Cells with, or without NAC, were treated with MLN4924 (0.33 μM), CQ (20 μM), or both for 4 hours. ROS generation was determined by H2-DCFDA staining and flow cytometry (**P < 0.01, n = 3). (B-C) Effect of NAC on mRNA (B) and protein (C) expression of NOXA was determined by Q-PCR and immunoblotting respectively (**P < 0.01, n = 3). (D-E) The antioxidant NAC rescued cell viability and diminished drug-induced apoptosis. HepG2 and Huh7 cells were pre-treated with NAC (50 μM) for 2 hours, and then were treated with MLN4924 (0.33 μM), CQ (20 μM), or both for 36 hours. Apoptotic cells were determined by Annexin V-FITC/PI double-staining through FACS analysis (D). Cell viability was measured using ATPLite assay (E). (F) Effect of combined treatment of both NAC and siORC1/CDT1 on NOXA expression and apoptosis induction. The expression of CDT1, ORC1, c-Caspase3 and NOXA was determined by immunoblotting. Tubulin served as a loading control. All data are representative of at least three independent experiments.
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Figure 5: Production of excessive ROS contributes to MLN4924+CQ-induced NOXA transactivation and apoptotic induction of liver-cancer cells(A) Effect of MLN4924 or CQ alone or the MLN4924+CQ combination, on ROS generation. HepG2 and Huh7 cells were pre-treated with NAC (50 μM) for 2 hours. Cells with, or without NAC, were treated with MLN4924 (0.33 μM), CQ (20 μM), or both for 4 hours. ROS generation was determined by H2-DCFDA staining and flow cytometry (**P < 0.01, n = 3). (B-C) Effect of NAC on mRNA (B) and protein (C) expression of NOXA was determined by Q-PCR and immunoblotting respectively (**P < 0.01, n = 3). (D-E) The antioxidant NAC rescued cell viability and diminished drug-induced apoptosis. HepG2 and Huh7 cells were pre-treated with NAC (50 μM) for 2 hours, and then were treated with MLN4924 (0.33 μM), CQ (20 μM), or both for 36 hours. Apoptotic cells were determined by Annexin V-FITC/PI double-staining through FACS analysis (D). Cell viability was measured using ATPLite assay (E). (F) Effect of combined treatment of both NAC and siORC1/CDT1 on NOXA expression and apoptosis induction. The expression of CDT1, ORC1, c-Caspase3 and NOXA was determined by immunoblotting. Tubulin served as a loading control. All data are representative of at least three independent experiments.

Mentions: NOXA-dependent apoptosis can be partially, but not completely abrogated, by the blockage of DNA damage (Figure 4). To further elucidate the underlying mechanisms of MLN4924+CQ-induced NOXA expression, we investigated the potential role of ROS in NOXA expression, since ROS production during cellular stresses could trigger NOXA expression [36, 37] and MLN4924 induced elevation of ROS generation [20]. We first assessed the impact of drug treatment on mitochondrial transmembrane potential since the impaired mitochondrial electron transport chain serves as a major source of ROS production [26]. As shown in Suppl. Figure 4, treatment of cells with MLN4924+CQ caused significantly enhancement of mitochondrial depolarization when compared to MLN4924 or CQ treatment alone (Suppl. Figure 4). Next, ROS production was monitored with the cell permeable ROS indicator, 2′, 7′-dichlorodihydrofluorescein diacetate (H2-DCFDA), using flow-cytometry analysis [26]. MLN4924 or CQ alone increased the level of ROS while MLN4924+CQ significantly amplified the production of ROS compared to treatment alone (Figure 5A left panel). Moreover, the enhanced production of ROS induced by MLN4924+CQ was completely blocked by pre-treatment of cells with NAC, a classical ROS scavenger [36, 37] (Figure 5A right panel), which significantly down-regulated NOXA expression at both mRNA and protein level (Figure 5B and 5C). And reduction of ROS by NAC also attenuated apoptosis and reduced cell death (Figure 5D and 5E). These results demonstrated that enhanced production of ROS contributed to the induction of NOXA and apoptosis upon MLN4924+CQ treatment in liver cancer cells.


Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer.

Chen P, Hu T, Liang Y, Jiang Y, Pan Y, Li C, Zhang P, Wei D, Li P, Jeong LS, Chu Y, Qi H, Yang M, Hoffman RM, Dong Z, Jia L - Oncotarget (2015)

Production of excessive ROS contributes to MLN4924+CQ-induced NOXA transactivation and apoptotic induction of liver-cancer cells(A) Effect of MLN4924 or CQ alone or the MLN4924+CQ combination, on ROS generation. HepG2 and Huh7 cells were pre-treated with NAC (50 μM) for 2 hours. Cells with, or without NAC, were treated with MLN4924 (0.33 μM), CQ (20 μM), or both for 4 hours. ROS generation was determined by H2-DCFDA staining and flow cytometry (**P < 0.01, n = 3). (B-C) Effect of NAC on mRNA (B) and protein (C) expression of NOXA was determined by Q-PCR and immunoblotting respectively (**P < 0.01, n = 3). (D-E) The antioxidant NAC rescued cell viability and diminished drug-induced apoptosis. HepG2 and Huh7 cells were pre-treated with NAC (50 μM) for 2 hours, and then were treated with MLN4924 (0.33 μM), CQ (20 μM), or both for 36 hours. Apoptotic cells were determined by Annexin V-FITC/PI double-staining through FACS analysis (D). Cell viability was measured using ATPLite assay (E). (F) Effect of combined treatment of both NAC and siORC1/CDT1 on NOXA expression and apoptosis induction. The expression of CDT1, ORC1, c-Caspase3 and NOXA was determined by immunoblotting. Tubulin served as a loading control. All data are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496198&req=5

Figure 5: Production of excessive ROS contributes to MLN4924+CQ-induced NOXA transactivation and apoptotic induction of liver-cancer cells(A) Effect of MLN4924 or CQ alone or the MLN4924+CQ combination, on ROS generation. HepG2 and Huh7 cells were pre-treated with NAC (50 μM) for 2 hours. Cells with, or without NAC, were treated with MLN4924 (0.33 μM), CQ (20 μM), or both for 4 hours. ROS generation was determined by H2-DCFDA staining and flow cytometry (**P < 0.01, n = 3). (B-C) Effect of NAC on mRNA (B) and protein (C) expression of NOXA was determined by Q-PCR and immunoblotting respectively (**P < 0.01, n = 3). (D-E) The antioxidant NAC rescued cell viability and diminished drug-induced apoptosis. HepG2 and Huh7 cells were pre-treated with NAC (50 μM) for 2 hours, and then were treated with MLN4924 (0.33 μM), CQ (20 μM), or both for 36 hours. Apoptotic cells were determined by Annexin V-FITC/PI double-staining through FACS analysis (D). Cell viability was measured using ATPLite assay (E). (F) Effect of combined treatment of both NAC and siORC1/CDT1 on NOXA expression and apoptosis induction. The expression of CDT1, ORC1, c-Caspase3 and NOXA was determined by immunoblotting. Tubulin served as a loading control. All data are representative of at least three independent experiments.
Mentions: NOXA-dependent apoptosis can be partially, but not completely abrogated, by the blockage of DNA damage (Figure 4). To further elucidate the underlying mechanisms of MLN4924+CQ-induced NOXA expression, we investigated the potential role of ROS in NOXA expression, since ROS production during cellular stresses could trigger NOXA expression [36, 37] and MLN4924 induced elevation of ROS generation [20]. We first assessed the impact of drug treatment on mitochondrial transmembrane potential since the impaired mitochondrial electron transport chain serves as a major source of ROS production [26]. As shown in Suppl. Figure 4, treatment of cells with MLN4924+CQ caused significantly enhancement of mitochondrial depolarization when compared to MLN4924 or CQ treatment alone (Suppl. Figure 4). Next, ROS production was monitored with the cell permeable ROS indicator, 2′, 7′-dichlorodihydrofluorescein diacetate (H2-DCFDA), using flow-cytometry analysis [26]. MLN4924 or CQ alone increased the level of ROS while MLN4924+CQ significantly amplified the production of ROS compared to treatment alone (Figure 5A left panel). Moreover, the enhanced production of ROS induced by MLN4924+CQ was completely blocked by pre-treatment of cells with NAC, a classical ROS scavenger [36, 37] (Figure 5A right panel), which significantly down-regulated NOXA expression at both mRNA and protein level (Figure 5B and 5C). And reduction of ROS by NAC also attenuated apoptosis and reduced cell death (Figure 5D and 5E). These results demonstrated that enhanced production of ROS contributed to the induction of NOXA and apoptosis upon MLN4924+CQ treatment in liver cancer cells.

Bottom Line: However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance.Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins.Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT
Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.

No MeSH data available.


Related in: MedlinePlus